(NA+ + K+) Adenosine triphosphatase is the enzyme in the plasma membrane of all animal cells that is responsible for the active transport of sodium out of and potassium into the cytoplasm. It is also the primary target of the cardiotonic steroids such as digitalis and digoxin. It is our objective to obtain extensive information about its molecular structure and use this knowledge to gain insight into the mechanism of active transport. It has been demonstrated that the functional subunit, Alpha, is very large (1100 residues) and spans the plasma membrane, but more detailed information is lacking. Vesicles of plasma membrane, containing high concentrations of (Na+ + K+)-ATPase and all sealed in a right-side-out orientation, will be modified covalently with isethionyl 3-(N-2,4-dinitrophenyl)-3-aminopropionimdate, an impermeant reagent that reacts with the exposed lysine residues of the protein. The lysines on the extracytoplasmic surface of the enzyme that react with the reagent will be identified by isolating and sequencing the peptides in which they are located. The binding site for the cardiotonic steroids is also located on this extracytoplasmic surface, and affinity reagents derived from these drugs and specific for the modification of this site are available. They will be used to identify the portions of the amino acid sequence of this protein that form this site. Finally, by exploiting changes that occur in the reactivity of specific sulfhydryl residues in the Alpha chain during the changes in conformation, we will be able to identify those regions in the protein involved in these conformational changes. The location of these sulfhydryls in the overall sequence of the protein will be determined. All of these experiments will increase our understanding of (Na+ + K+) adenosine triphosphatase whose function is of central importance to those physiological mechanisms which pace heartbeat and create the action potentials of the nervous system.